CN110243790A - The code-shaped fluorescent bio-probes of the gene of a kind of pair of hydrogen peroxide specificly-response and its construction method and purposes - Google Patents
The code-shaped fluorescent bio-probes of the gene of a kind of pair of hydrogen peroxide specificly-response and its construction method and purposes Download PDFInfo
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- CN110243790A CN110243790A CN201910260413.6A CN201910260413A CN110243790A CN 110243790 A CN110243790 A CN 110243790A CN 201910260413 A CN201910260413 A CN 201910260413A CN 110243790 A CN110243790 A CN 110243790A
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the code-shaped fluorescent bio-probes of the gene of a kind of pair of hydrogen peroxide specificly-response and its construction method and purposes, and having the code-shaped fluorescence probe of gene of specificly-response to hydrogen peroxide is made up of the both ends that the PrxA and TrxA sensitive to hydrogen peroxide specificity are fused to cpYFP fluorescin respectively.Fluorescence probe of the present invention has high sensitivity and selectivity to hydrogen peroxide, intracellular and extracellular equal can respond rapidly to hydrogen peroxide, especially the probe is a kind of code-shaped probe of gene, it can make up type expression, it realizes the variation of Real-time and Dynamic Detection concentration of hydrogen peroxide intracellular and is used for cell imaging, have broad application prospects for research redox metabolism associated signal paths.
Description
Technical field
The invention belongs to the method fields more particularly to a kind of pair of peroxidating of fluorescent bio-probes and its detection hydrogen peroxide
The code-shaped fluorescent bio-probes of the gene of hydrogen specificly-response and its construction method and purposes.
Background technique
Active oxygen (ROS) was once considered being lethal to cell, but was presently considered to be a kind of and participates in a variety of oxidations also
The molecule of original signal conduction, the generation of normal function and disease to cell contribute.Low-level ROS intracellular being capable of conduct
Signaling molecule.When oxidation resistance range of the ROS content of generation intracellular far more than a series of antioxidant systems intracellular, ROS
Level can further increase.The latter is related to the induction of oxidative stress and relevant signal transduction, and it is characterized in that ROS
The variation of the cellular redox stable state of induction and/or destruction to biomolecule (such as DNA, albumen and lipid).
Because the generation and removing of ROS are related to complicated mechanism, and can have really specificity to the active oxygen of a certain seed type
Report molecule shortage, the level of the ROS in quantitative intracellular and subcellular structure is a challenging job.
A main component hydrogen peroxide of ROS is considered as aerobe inevitably but is not phase for many years
The by-product of prestige, if consumption is faster more advantageous to cell.But it is more emphasized recently by major seminar, mammal
A variety of different physiological reactions, such as cell Proliferation, differentiation and migration etc. can be mediated by generating hydrogen peroxide.This process
Mean " redox " in cell normal processes and progression of disease, such as angiogenesis, oxidative stress and aging and cancer
Signal function is played in the process.A kind of hydrogen peroxide detection method of simple high sensitivity has extensive analysis application value.
Though in monitoring food, the consumer goods and ambient water, such as rainwater, in low-level hydrogen peroxide, or in detection aquae hydrogenii dioxidi
It puts down to study the generation aspect whether it is used as signal transduction molecule still to induce many diseases and all there is consequence.
Fluorescence probe is a foundation stone of active somatic cell real time imagery and is one in cell biology strong
Tool.The probe encoded derived from the gene of fluorescin is observed and is tracked just to play in various process intracellular and lead revolution in real time
Effect.Nearest some progress, including " passive " label for measuring the expression and positioning of biomolecule in active somatic cell
The continuous development of object, and what is developed monitor more complicated process intracellular, such as the dynamic variation process of small molecule courier, protease
Interaction process etc. between activation and protein-protein, the emergence of indicator have all pushed the cell of fluorescin raw
Development in terms of object.
In order in this important signaling molecule of detection hydrogen peroxide intracellular, the code-shaped probe of the gene of relevant report is also
Through being developed, but it is in response to more quick, sensitiveer and more accurate bioprobe and need to be developed, and then more
Deficiency existing for existing hydrogen peroxide detection method is mended, and to research, physiology and the pathology for pushing redox signal transduction
Research be of great significance.
Summary of the invention
The purpose of the present invention is to the deficiencies of existing hydrogen peroxide detection method, are constructed by the means of Gene Fusion
It is a kind of to have high sensitivity and the highly selective code-shaped fluorescent bio-probes of gene to hydrogen peroxide in vivo, in vitro
Construction method.
Second object of the present invention is that the code-shaped fluorescence of the gene for providing a kind of pair of hydrogen peroxide specificly-response is raw
Physical prospecting needle.
Third object of the present invention is to provide a kind of polynucleotide molecule.
Fourth object of the present invention is to provide a kind of recombinant plasmid.
Of the invention the 5th, which is designed to provide, visits the code-shaped biological of the gene of hydrogen peroxide specificly-response
The application of needle.
To achieve the goals above, the present invention provides the code-shaped fluorescence of the gene of a kind of pair of hydrogen peroxide specificly-response
The construction method of bioprobe, which is characterized in that the construction method be by the sensitive PrxA of hydrogen peroxide specificity and
TrxA is fused to the both ends of cpYFP fluorescin respectively.
As a preferred embodiment, the cysteine of TrxA amino acid sequence is first replaced with into polar amino acid, is obtained
TrxA mutant is merged again.
As a preferred embodiment, the polar amino acid includes glutamine, asparagine, serine, tyrosine,
Arginine, histidine, lysine, any one in asparatate and glutamic acid.
As a preferred embodiment, the construction method is comprised the following specific steps that:
Step 1: the 39th cysteine of TrxA amino acid sequence shown in SEQ ID NO.1 is replaced with into Polar Amides
Acid obtains TrxA mutant；
Step 2: by TrxA mutant obtained in the nucleic acid sequence, step 1 of coding PrxA shown in SEQ ID NO.2
The nucleic acid sequence of coding cpYFP shown in nucleic acid sequence and SEQ ID NO.3 is merged, and hydrogen peroxide specificly-response is obtained
The code-shaped fluorescent bio-probes of gene.
As a preferred embodiment, TrxA mutant is located at the nitrogen end or carbon teminal of cpYFP, centre insertion link peptide X1, institute
Stating X1 is GGGS, GGGGS, SAG, GT, LAEAAAKEAA, any one in TSGSPGLQEFGT.
As a preferred embodiment, PrxA is located at the carbon teminal or nitrogen end of cpYFP, and centre insertion link peptide X2, the X2 are
Any one in GGGS, GGGGS, SAG, GT, LAEAAAKEAA, TSGSPGLQEFGT.
In order to realize second purpose of the invention, the present invention provides using above-mentioned construction method prepare to peroxide
Change the code-shaped fluorescent bio-probes of gene of hydrogen specificly-response.
In order to realize third purpose of the present invention, the present invention provides a kind of polynucleotide molecules, which is characterized in that described
The nucleic acid encode of polynucleotide molecule is above-mentioned to the code-shaped fluorescent bio-probes of the gene of hydrogen peroxide specificly-response.
In order to realize the 4th purpose of the invention, the present invention provides a kind of recombinant plasmids, which is characterized in that the recombination
Plasmid expression vector includes to encode above-mentioned polynucleotide molecule gene.
In order to realize the 5th purpose of the invention, the present invention provides the above-mentioned gene volumes to hydrogen peroxide specificly-response
Application of the pattern fluorescent bio-probes in the detection of hydrogen peroxide or organic peroxide.
The side of external hydrogen peroxide qualitatively or quantitatively detected is carried out using the code-shaped fluorescent bio-probes of gene of the present invention
Method is, by the gene cloning to expression vector of above-mentioned coding fluorescence bioprobe, to be transformed into E. coli DH5 α
In, screening positive clone, and positive recombinant plasmid is extracted, it is transformed into expression bacterial strain E.coli BL21 (DE3), protein expression
After purified.The albumen of purifying is diluted to a certain concentration detection hydrogen peroxide.
The code-shaped fluorescent bio-probes of gene carry out the qualitative or quantitative detection of external hydrogen peroxide specifically comprising following excellent
Select mode:
(1) expression vector of the fluorescent bio-probes is pET28a.
(2) condition of the fluorescent bio-probes protein expression is that expression bacterial strain is cultivated at 37 DEG C, and concentration reaches OD600?
When 0.4-0.6 or so, addition 0.1-0.3mM IPTG is induced, and the temperature for expressing albumen is 16-20 DEG C, and expression time is
15-24h。
(3) condition of the fluorescent bio-probes protein purification is to utilize the buffer solution A for being added with 2-10mM mercaptoethanol
(20mM NaH2PO4, 0.5M NaCl, pH7.0) and suspension cell, ultrasonication, 9000-12000g centrifugation 10-20 minutes, supernatant
Purify by 0.45 μm of filter, then with nickel column.Eluent is the buffer solution B added with 2-10mM mercaptoethanol
(20mM NaH2PO4, 0.5M NaCl, 0.5M imidazoles, pH7.0).Albumen after purification carries out ultrafiltration using 15mL super filter tube, surpasses
Filter revolving speed is 4680~5000rpm.Finally albumen ultrafiltration is delayed except imidazoles is stored in the 50mM sodium phosphate containing 1~2mM DTT
In fliud flushing (pH7.0).
(4) fluorescent bio-probes of vitro detection hydrogen peroxide are in 50mM sodium phosphate buffer (pH7.0),
Under 530nm excitation, there are two excitation peaks at 410-420nm and 495-505nm.Under 485nm shooting condition, in 515-
There is excitation peak at 525nm.
(5) concentration of the fluorescent bio-probes of vitro detection hydrogen peroxide is 0.4-1 μM.
(6) detection of vitro detection hydrogen peroxide is limited to 0.1-1 μM.
(7) have to the response of hydrogen peroxide highly selective.
The side of internal hydrogen peroxide qualitatively or quantitatively detected is carried out using the code-shaped fluorescent bio-probes of gene of the present invention
Method.Expression the code-shaped fluorescent bio-probes of said gene E. coli BL21 (DE3) be centrifuged, wash after dilute
Hydrogen peroxide detection is carried out in detection buffer.
The code-shaped fluorescent bio-probes of gene carry out the qualitative or quantitative detection of internal hydrogen peroxide specifically comprising following excellent
Select mode:
(1) concentration is in OD after expressing the Escherichia coli dilution of the code-shaped fluorescent bio-probes of said gene600It is 0.1~0.4
Between.
(2) the Escherichia coli washing and dilution for expressing the code-shaped fluorescent bio-probes of said gene are 50mM phosphoric acid
Sodium buffer (pH7.0)
(3) Escherichia coli for expressing the code-shaped fluorescent bio-probes of said gene are enable to respond quickly the peroxide of external source addition
Change hydrogen, the time is no more than 30s.
(4) the code-shaped fluorescent bio-probes of said gene can be used for active somatic cell imaging in vivo.
(5) inspection of the hydrogen peroxide of the Escherichia coli response external source addition of the code-shaped fluorescent bio-probes of said gene is expressed
Survey is limited to 0.5-5 μM.
It is an advantage of the current invention that the construction method of such probe molecule provides a kind of building response in (1) present invention
The method and thinking of the code-shaped fluorescence probe of the gene of different substrates.(2) such fluorescent bio-probes is in intracellular and extracellular equal energy
Rapidly, highly sensitive and respond hydrogen peroxide with high selectivity, and the detection limit of the hydrogen peroxide in response external source addition intracellular is bright
The aobvious detection lower than in relation to research report under equal conditions limits, and is that an overdelicate hydrogen peroxide response type biological is visited
Needle.(3) such fluorescent bio-probes can be used in the cell imaging of Escherichia coli response hydrogen peroxide.(4) no matter being gone back extracellular
It is intracellular, such fluorescent bio-probes is to the quick response of hydrogen peroxide, high sensitivity and highly selective can greatly push away
The development of the research for the signal path that dynamic hydrogen peroxide participates in establishes base for the research of the related physiology of redox and pathology
Plinth.
Detailed description of the invention
When Fig. 1 is that the code-shaped fluorescent bio-probes of gene of the present invention of purifying respond various concentration hydrogen peroxide, fluorescence Spectra
The variation of figure, abscissa are the wavelength (nm) of exciting light, and ordinate is to be carried out with the fluorescence of the above-mentioned fluorescent bio-probes of reduced form
The fluorescence intensity of Regularization.It is the detection of 530nm condition in launch wavelength.Above-mentioned fluorescent bio-probes albumen is in response hydrogen peroxide
Afterwards, peak value can decline at 415nm, and peak value can rise at 500nm, with the difference of concentration of hydrogen peroxide, at 415nm and 500nm
Peak change degree is different, i.e. F500nm/F415nmRatio variation is different.
Fig. 2 is that the code-shaped fluorescent bio-probes of gene of the present invention of purifying respond the change in fluorescence of different oxidants, horizontal seat
Different oxidation materials are designated as, ordinate is oxidized ratio R (F500nm/F415nm) divided by reduced form ratio R0Value.It aoxidizes
Afterwards, R/R0Increase, degree of oxidation is bigger, R/R0It is bigger, if being not responding to R/R0It is then 1.Wherein added oxide is respectively 10
μM hydrogen peroxide, 1 μM of tert-butyl hydroperoxide, 1mM oxidized form of glutathione, 40 μM of 1- (hydroxy-n NO- nitrogen oxygroup)-L- dried meat
(SIN-1, peroxidating are sub- for propylhomoserin disodium salt (nitric oxide donors), 10 μM of cysteines and 10 μM of morpholinyl-Si De imines
Nitrate donor).
Fig. 3 be express fluorescent bio-probes of the present invention Escherichia coli response hydrogen peroxide before (Fig. 3 A) and afterwards (Fig. 3 B) exist
The cell imaging figure detected under 488nm lasing condition.
Fig. 4 be fluorescent bio-probes of the present invention Escherichia coli it is intracellular respond various concentration hydrogen peroxide the case where.Figure
4A is the R/R for expressing the Escherichia coli of fluorescent bio-probes of the present invention under the stimulation of 0.5 μM and 5 μM hydrogen peroxide0At any time
Variation diagram, abscissa are the time, and ordinate is oxidized ratio R (F500nm/F415nm) divided by reduced form ratio R0Value.Fig. 4 B is
Express R/R of the Escherichia coli of fluorescent bio-probes of the present invention under the stimulation of various concentration hydrogen peroxide0Variation column
Figure, abscissa are the concentration of hydrogen peroxide, and ordinate is oxidized ratio R (F500nm/F415nm) divided by reduced form ratio R0Value.
Specific embodiment
Hereinafter, technology of the invention is described in detail in conjunction with specific embodiment.It is appreciated that the various in detail below
Embodiment is only used for helping skilled in the art to understand the present invention, rather than limitation of the present invention.
Material source in the following example are as follows:
Plasmid vector pET28a is purchased from Novagen company.
Restriction enzyme EcoR I, Hind III and primeSTAR are the commercial product of Takara company.
Multiple clips homologous recombination one-step cloning kit is purchased from Vazyme company.
CpYFP gene order is synthesized by Shanghai Rui Di biotechnology company and is connected to the BamH I/Xho I enzyme of pET28a
Among enzyme site.Plasmid with PrxA and TrxA segment is constructed by this laboratory and respectively as PrxA and TrxA gene magnification
Template.
Unless otherwise indicated, the specific experiment in the following example is carried out according to conventional method in that art and condition, or is abided by
According to the product manual of kit.Quantitative experiment in following case study on implementation is respectively provided with and repeats to test three times, and result is average value
± standard deviation.
The building of the 1 code-shaped fluorescent bio-probes of hydrogen peroxide specificly-response gene of embodiment
1, the amplification of TrxA (C39E) genetic fragment
Stable disulfide bond is formed between PrxA and TrxA to need the C of TrxAP(i.e. 39 cysteines) mutation is fallen.This
Experiment, which is selected, sports glutamic acid for TrxA39 cysteines.Design upstream primer TrxA-C39E-F:5 '-GACATGGTGC
GGTCCCGAAAAGGCCATTGCGC-3 ' and downstream primer TrxA-C39E-R:5 '-TGGGCGCAATGGCCTTTTCGGGACCG
CACCATGTCG-3 ', the plasmid with TrxA gene using the building of this laboratory are that template carries out rite-directed mutagenesis, and screens
Success mutant recombinant plasmid, wherein sequence shown in underscore is mutational site.Using the mutant recombinant plasmid of generation as template,
Design upstream primer TrxA-F:5 '-ATGGGTGCCTCTGAACACGTCC-3 ' and downstream primer TrxA-pET28a-R: By TrxA (C39E) gene
Fragment amplification comes out, and wherein thickened portion is the homologous arm section homologous with pET28a plasmid vector, and part shown in underscore is
HindIII restriction enzyme site.PCR product carries out purification and recovery.
2, the amplification of PrxA and cpYFP genetic fragment
It is with the plasmid with cpYFP gene synthesized using the recombinant plasmid with PrxA with the building of this laboratory
Template separately designs primer and comes out PrxA and cpYFP gene fragment amplification.Design upstream primer pET28a-PrxA-F:
With downstream primer PrxA-R:5 '-CAGGTGCTTGATGACAGTCTCGG-3 ' carry out PrxA amplification, wherein thickened portion be with
The homologous homologous arm section of pET28a plasmid vector, sequence shown in underscore are EcoRI restriction enzyme site.Design upstream primer
PrxA-X1-cpYFP-F:
TACAACAGCCACAACGTCTATATC-3 ' and downstream primer cpYFP-X2-TrxA-R: Carry out the expansion of cpYFP
Increase.Wherein upstream primer thickened portion is the homologous arm section homologous with PrxA, and sequence shown in underscore is that X1 link peptide is SAG
Coded sequence.Downstream primer thickened portion is the homologous arm section homologous with TrxA, and sequence shown in underscore is X2 link peptide
For the coded sequence of GT.PCR product carries out purification and recovery.
3, the building of the building of the code-shaped fluorescent bio-probes of hydrogen peroxide specificly-response gene and recombinant plasmid
Double digestion, digestion products purification and recovery are carried out to pET28a plasmid using EcoR I and Hind III.It will be above-mentioned pure
The pET28a carrier of TrxA, PrxA and cpYFP segment and digestion of changing recycling prepares linked system according to such as table one on ice bath.
The preparation of one multiple clips one-step cloning of table connection reaction solution
Wherein:
The amount of X/Y is according to formula:
The most suitable usage amount of each segment=[0.02 × segment base number] ng (0.03pmol)
It is calculated.
After above-mentioned reaction system is mixed gently using liquid-transfering gun, 30min is reacted in 37 DEG C of thermostat water baths, it is cold on ice immediately
But, the above-mentioned reaction system mixed liquor of 10 μ L is taken to be transformed into DH5 α competence bacterial strain, picking individual colonies carry out positive colony screening
And nucleic acid sequencing validation.The plasmid being proved to be successful is to have to the code-shaped biological of hydrogen peroxide specificly-response gene
The recombinant plasmid of probe.
The expression and purifying of the 2 code-shaped fluorescent bio-probes of hydrogen peroxide specificly-response gene of embodiment
Take 1uL that the correctly above-mentioned recombination matter to the code-shaped fluorescent bio-probes of hydrogen peroxide specificly-response gene is sequenced
The method that grain is converted by competence is transformed into expression bacterial strain E.coil BL21 (DE3), carries out the expression of each recombinant protein.
Specific expression step are as follows:
1, picking conversion is in the LB (Kan of expression bacterial strain E.coil BL21 (DE3)+Resistance) single colonie on plate, inoculation
To containing 100 μ g/ μ L Kan+5mL liquid LB test tube in, 37 DEG C, 200rpm overnight incubation.
2, according to 1% inoculum concentration, the bacterium solution cultivated before 1mL is added to the triangular flask of LB culture medium of the 250mL containing 100mL
In, final concentration of 100 μ g/ μ L Kan is added+, 37 DEG C, 200rpm, shaken cultivation 2-3 hours, until the OD value of bacterium solution reaches
0.4-0.6。
3, addition 0.1mM IPTG inducible protein expression, protein expression temperature are 20 DEG C, and 220rpm cultivates 14-20h.
4, after cultivating, bacterium solution a part is poured into 50mL centrifuge tube, and 10000rpm is centrifuged 15 minutes collection thallus.One
Response of the fresh bacterium solution in part for hydrogen peroxide measures.
The above-mentioned protein purification steps to the code-shaped fluorescent bio-probes of hydrogen peroxide specificly-response gene specifically:
1, bacterial cell disruption: buffer solution A (the 20mM NaH added with 5mM mercaptoethanol is utilized2PO4, 0.5M NaCl, pH7.0)
The thallus for being collected in centrifugation bottom of the tube is suspended into (100mL thallus is suspended in 20-30mL buffer).High pressure is crushed in advance even
Pulp grinder is cooled to 4 DEG C in advance, removes 20% ethyl alcohol of preservation, with deionized water cleaning robot 1-2 times, then with the buffering for dissolving thallus
Liquid cleaning robot 1-2 times.The pressure of machine is adjusted to 1000bar or so, loading collects broken liquid with clean centrifuge tube, then
Repetition is added in specimen cup, repeats to be crushed 2-3 times, until solution becomes sticky clarification.
2, it bacterial sediment: is put into collecting in the centrifuge tube for having the liquid being crushed in 4 DEG C of pre-cooling centrifuges, 12000rpm
It is centrifuged 15min, makes bacterial sediment in centrifugation bottom of the tube, supernatant is poured into clean centrifuge tube.
3, it filters: filtering supernatant using 0.45 μm of filter, prevent impurity from blocking pillar.
4, upper prop and purifying: above-mentioned fluorescent bio-probes albumen is yellow, directly can carry out albumen with the self-chambering column of nickel
Purifying.Before loading, buffer solution A (20mM NaH is utilized2PO4, 0.5M NaCl, pH7.0) pillar is balanced.Supernatant is direct
It is added in self-chambering column, using the effect of gravitational settling, flows through self-chambering column, combine albumen and nickel column.Buffer solution A is used again
(20mM NaH2PO4, 0.5M NaCl, pH7.0) impurity protein not with nickel ion specific binding is washed.Again directly
With 100% buffer solution B (20mM NaH2PO4, 0.5M NaCl, 0.5M imidazoles, pH7.0) and it is eluted, collect coloured wash
De- liquid.
5, protein concentration: the albumen being purified pours into the super filter tube that 15mL molecular cut off is 10kDa, with containing
The sodium phosphate buffer (pH7.0) of 1mM DTT is retained in the albumen for finally removing imidazoles containing DTT's as dilution cleaning solution
In phosphate buffer (pH7.0).If (saving albumen, adding glycerol, make final glycerol concentration 20%, -20 DEG C of preservations.)
The 3 code-shaped fluorescent bio-probes of hydrogen peroxide specificly-response gene of embodiment respond hydrogen peroxide detection in vitro
First by mother liquid concentration be 10M hydrogen peroxide with 10 times of extension rate, be diluted to respectively concentration be 1M,
100mM, 10mM, 1mM, 100 μM, 10 μM of hydrogen peroxide.It is ready-to-use every time before experiment since hydrogen peroxide is unstable.Benefit
With the concentration of the above-mentioned fluorescent bio-probes albumen of nucleic acid/Protein Detection instrument measurement purifying concentration.Because being guarantor after protein purification
Its reproducibility is held, is stored in the solution containing DTT.DTT is gone out using a P6 desalting column of Bio-Rad before experiment every time
It goes.It is used before each extracellular experiment, it is current that albumen now removes DTT.Measure above-mentioned fluorescent bio-probes albumen response hydrogen peroxide
Experiment in, the above-mentioned fluorescent bio-probes albumen of concentration is added to the ratio of the phosphate buffer (pH 7.0) added with 1mL 50mM
In color ware, final concentration is diluted to 1 μM or so.Hitachi's F-4600 sepectrophotofluorometer is opened, cuvette is put into, utilizes Hitachi
Under the conditions of F-4600 fluophotometer record transmitting light is 530nm, the fluorescence intensity of exciting light in 380nm to 515nm range.Again
Exciting light is recorded to emit the fluorescence intensity of light in 505nm to 600nm range, as the background of experiment under the conditions of 485nm.It
Afterwards, into cuvette add various concentration hydrogen peroxide, re-record with exciting light under background equal conditions and emit light it is glimmering
Luminous intensity.The slit of excitation and transmitting light is 5nm, and the speed of length scanning is 2400nm/min, photomultiplier tube (PMT) electricity
Pressure is 700V, and the control group of experiment is the deionized water for adding equal volume.Attached drawing 1 shows the above-mentioned of vitro detection hydrogen peroxide
Fluorescent bio-probes are in 50mM sodium phosphate buffer (pH7.0), under 530nm excitation, in 410-420nm and 495-505nm
There are two excitation peaks at place.Under 485nm shooting condition, there is excitation peak at 515-525nm, the detection for responding hydrogen peroxide is limited to
0.1-1μM.After different oxidation materials is configured to your corresponding concentration, according to trans- addition identical with hydrogen peroxide and survey
The selectivity of fixed above-mentioned fluorescent bio-probes.Attached drawing 2 shows above-mentioned fluorescent bio-probes in addition to response hydrogen peroxide and organic mistake
(natural substrate of Prx is both lived) except oxide to be not responding to the oxide of other tests, illustrates above-mentioned biological
Probe has the response of hydrogen peroxide highly selective.
The living body of response hydrogen peroxide in the 4 code-shaped fluorescent bio-probes body of hydrogen peroxide specificly-response gene of embodiment
Cell imaging
After fresh bacterium solution expression, 5mL or so bacterium solution is taken to be centrifuged, culture medium is removed in centrifugation.Utilize 7.0 phosphoric acid buffer of pH
Liquid washes twice, and is suspended in same buffer.The final concentration of OD of somatic cells suspension600Value is 1.26.Fluorescence is clapped
It is Nikon Eclipse Ti-E micrometron, Photomertrics Evolve 512EMCCD and high stable according to what is used
The Sutter Lambda XL light source of type.What is used is the oil mirror of 100 × 1.4NA of a Plan Apo VC.Fluorescence is inverted aobvious
In micro mirror shooting, on 3 μ L cell suspending liquids of addition and glass slide, then covered with coverslip, upside down is on objective table.Benefit
It with 488nm exciting light, is detected within the scope of 500-550nm, time for exposure 1000ms.Then glass slide is stretched out from coverslip
One jiao of dropwise addition 4 μ L, 10 μM of hydrogen peroxide.Then it detects and takes pictures under identical condition.Attached drawing 3A, which is shown, to be not added with
The colorectal cell figure of the above-mentioned fluorescent bio-probes of expression of hydrogen oxide.Attached drawing 3B is shown in after response hydrogen peroxide, and 488nm swashs
Under shining, the fluorescence of cell is remarkably reinforced.Above-mentioned fluorescent bio-probes can be used in active somatic cell imaging.
The detection of response hydrogen peroxide in the 5 code-shaped fluorescent bio-probes body of hydrogen peroxide specificly-response gene of embodiment
First by mother liquid concentration be 10M hydrogen peroxide with 10 times of extension rate, be diluted to respectively concentration be 1M,
100mM, 10mM, 1mM, 100 μM, 10 μM of hydrogen peroxide.It is ready-to-use every time before experiment since hydrogen peroxide is unstable.Newly
Thalline were collected by centrifugation for fresh thallus culture medium, and washs 1-2 times in the phosphate buffer of pH7.0, is resuspended in same
In buffer.The diluted thallus suspension liquid of 8-15 μ L is added in experiment in the cuvette containing the same phosphate buffer of 1mL
It is measured.Hitachi's F-4600 sepectrophotofluorometer is opened, cuvette is put into, is remembered using Hitachi's F-4600 fluophotometer
Under the conditions of record transmitting light is 530nm, the fluorescence intensity of exciting light in 380nm to 515nm range.Re-recording exciting light is 485nm
Under the conditions of, the fluorescence intensity of transmitting light in 505nm to 600nm range, as the background of experiment.Later, it is added into cuvette
The hydrogen peroxide of various concentration re-records with exciting light under background equal conditions and emits the fluorescence intensity of light.Excitation and transmitting
The slit of light is 5nm, and the speed of length scanning is 2400nm/min, and photomultiplier tube (PMT) voltage is 700V, pair of experiment
It is the deionized water of addition equal volume according to group.The Escherichia coli of above-mentioned fluorescent bio-probes albumen are expressed in attached drawing 4A display
E.coil BL21 (DE3) can reach no matter the hydrogen peroxide of response low concentration kills the hydrogen peroxide of high concentration in 30s
Stablize, above-mentioned fluorescent bio-probes energy quick response hydrogen peroxide.Attached drawing 4B is shown, measures the peroxide of 0.2-30 μM of concentration range
Change hydrogen, the minimum of probe response hydrogen peroxide is limited to 0.5 μM, and after concentration is more than 5 μM, the fluorescence of probe just no longer changes,
Illustrate that the detection of above-mentioned probe response hydrogen peroxide intracellular is limited to 0.5-5 μM, can believe well using redox in vivo
In the research of number access, lay the foundation for the research of physiology and pathology.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
SEQUENCE LISTING
<110>East China University of Science
<120>the code-shaped fluorescent bio-probes of the gene of a kind of pair of hydrogen peroxide specificly-response and its construction method and purposes
<130> /
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 110
<212> PRT
<213> Aspergillus nidulans
<400> 1
Met Gly Ala Ser Glu His Val Pro Pro Ile Thr Ser Lys Ala Glu Phe
1 5 10 15
Gln Glu Lys Val Leu Asn Ala Lys Gly Phe Val Val Val Asp Cys Phe
20 25 30
Ala Thr Trp Cys Gly Pro Cys Lys Ala Ile Ala Pro Thr Val Glu Lys
35 40 45
Phe Ala Gln Thr Tyr Thr Asp Ala Ser Phe Tyr Gln Ile Asp Val Asp
50 55 60
Glu Leu Ser Glu Val Ala Ala Glu Leu Gly Ile Arg Ala Met Pro Thr
65 70 75 80
Phe Leu Leu Phe Lys Asp Gly Gln Lys Val Ser Asp Val Val Gly Ala
85 90 95
Asn Pro Gly Ala Leu Glu Ala Gly Ile Lys Ala Leu Leu Ala
100 105 110
<210> 2
<211> 507
<212> DNA
<213> Aspergillus nidulans
<400> 2
atgtctggac ttaaggccgg tgacagcttc cccgctgacg ttgtcttctc ctacattccc 60
tggactgagg agaagggcga gatcacctct tgcggtatcc ccattaacta ctacgcttcc 120
aaggagtggg ccgacaagaa ggtcatcctc ttcgccctcc ccggtgcctt cacccccgtc 180
tgctctgctc gccacgtccc tgagtacatc gagcgcctcc ccgagatccg cgccaagggt 240
gtcgatgttg ttgccgtcct tgcctacaac gatgctttcg tcatgagtgc ctggggtaag 300
gccaacggtg ttaagaacga cgacattctc ttcctttccg accccgaggc caagttctcc 360
aagagcatcg gctgggccga cgaggagggc cgtaccaagc gttatgccat cgtcctcgac 420
cacggcaagg tcacctacgc tgctctggag cccgccaaga accaccttga gttctccagc 480
gccgagactg tcatcaagca cctgtaa 507
<210> 3
<211> 741
<212> DNA
<213>artificial sequence
<400> 3
tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggccaac 60
ttcaagatcc gccacaacgt cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 120
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcttccag 180
tccgtcctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 240
accgccgccg ggatcactct cggcatggac gagctgtaca acgtggatgg cggtagcggt 300
ggcaccggca gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 360
gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 420
tacggcaagc tgaccctgaa gctgatctgc accaccggca agctgcccgt gccctggccc 480
accctcgtga ccaccctcgg ctacggcctg aagtgcttcg cccgctaccc cgaccacatg 540
aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 600
ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 660
ctggtgaacc gcatcgagct gaagggcatc ggcttcaagg aggacggcaa catcctgggg 720
cacaagctgg agtacaacta a 741
<210> 4
<211> 32
<212> DNA
<213>artificial sequence
<400> 4
gacatggtgc ggtcccgaaa aggccattgc gc 32
<210> 5
<211> 36
<212> DNA
<213>artificial sequence
<400> 5
tgggcgcaat ggccttttcg ggaccgcacc atgtcg 36
<210> 6
<211> 22
<212> DNA
<213>artificial sequence
<400> 6
atgggtgcct ctgaacacgt cc 22
<210> 7
<211> 47
<212> DNA
<213>artificial sequence
<400> 7
gctcgagtgc ggccgcaagc ttctaagcaa gcagagcctt gataccg 47
<210> 8
<211> 46
<212> DNA
<213>artificial sequence
<400> 8
caaatgggtc gcggatccga attcatgtct ggacttaagg ccggtg 46
<210> 9
<211> 23
<212> DNA
<213>artificial sequence
<400> 9
caggtgcttg atgacagtct cgg 23
<210> 10
<211> 54
<212> DNA
<213>artificial sequence
<400> 10
gagactgtca tcaagcacct gagcgccggc tacaacagcc acaacgtcta tatc 54
<210> 11
<211> 50
<212> DNA
<213>artificial sequence
<400> 11
ggacgtgttc agaggcaccc atggtgccgt tgtactccag cttgtgcccc 50
Claims (10)
1. the construction method of the code-shaped fluorescent bio-probes of the gene of a kind of pair of hydrogen peroxide specificly-response, which is characterized in that
The construction method is that the PrxA and TrxA sensitive to hydrogen peroxide specificity are fused to the two of cpYFP fluorescin respectively
End.
2. the structure of the code-shaped fluorescent bio-probes of gene of a kind of pair of hydrogen peroxide specificly-response according to claim 1
Construction method, which is characterized in that the cysteine of TrxA amino acid sequence is first replaced with into polar amino acid, obtains TrxA mutant
It is merged again.
3. according to right want 2 described in a kind of pair of hydrogen peroxide specificly-response the code-shaped fluorescent bio-probes of gene building
Method, which is characterized in that the polar amino acid includes glutamine, asparagine, serine, tyrosine, arginine, group
Propylhomoserin, lysine, any one in asparatate and glutamic acid.
4. the structure of the code-shaped fluorescent bio-probes of gene of a kind of pair of hydrogen peroxide specificly-response according to claim 3
Construction method, which is characterized in that the construction method comprises the following specific steps that:
Step 1: the 39th cysteine of TrxA amino acid sequence shown in SEQ ID NO.1 is replaced with into polar amino acid,
Obtain TrxA mutant；
Step 2: by the nucleic acid of TrxA mutant obtained in the nucleic acid sequence, step 1 of coding PrxA shown in SEQ ID NO.2
The nucleic acid sequence of coding cpYFP shown in sequence and SEQ ID NO.3 is merged, and the base of hydrogen peroxide specificly-response is obtained
Because of code-shaped fluorescent bio-probes.
5. the structure of the code-shaped fluorescent bio-probes of gene of a kind of pair of hydrogen peroxide specificly-response according to claim 4
Construction method, which is characterized in that TrxA mutant is located at the nitrogen end or carbon teminal of cpYFP, and centre insertion link peptide X1, the X1 are
Any one in GGGS, GGGGS, SAG, GT, LAEAAAKEAA, TSGSPGLQEFGT.
6. the structure of the code-shaped fluorescent bio-probes of gene of a kind of pair of hydrogen peroxide specificly-response according to claim 4
Construction method, which is characterized in that PrxA is located at the carbon teminal or nitrogen end of cpYFP, and centre insertion link peptide X2, the X2 is GGGS,
Any one in GGGGS, SAG, GT, LAEAAAKEAA, TSGSPGLQEFGT.
7. being compiled using the gene to hydrogen peroxide specificly-response that any construction method of claim 1-6 prepares
Pattern fluorescent bio-probes.
8. a kind of polynucleotide molecule, which is characterized in that the nucleic acid encode of the polynucleotide molecule is as claimed in claim 7
To the code-shaped fluorescent bio-probes of the gene of hydrogen peroxide specificly-response.
9. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid expression vector includes to encode multicore according to any one of claims 8
Thuja acid molecular gene.
10. it is as claimed in claim 7 to the code-shaped fluorescent bio-probes of the gene of hydrogen peroxide specificly-response in hydrogen peroxide
Or the application in the detection of organic peroxide.
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